Excess cholesterol induces mouse egg activation and
may cause female infertility
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Citation
Yesilaltay, Ayce, Gregoriy A. Dokshin, Dolores Busso, Li Wang,
Dalia Galiani, Tony Chavarria, Eliza Vasile, et al. “Excess
Cholesterol Induces Mouse Egg Activation and May Cause
Female Infertility.” Proceedings of the National Academy of
Sciences 111, no. 46 (November 3, 2014): E4972–E4980.
As Published
http://dx.doi.org/10.1073/pnas.1418954111
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National Academy of Sciences (U.S.)
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Final published version
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Wed May 25 22:42:17 EDT 2016
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http://hdl.handle.net/1721.1/96953
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Detailed Terms
Excess cholesterol induces mouse egg activation and
may cause female infertility
Ayce Yesilaltaya,1, Gregoriy A. Dokshina,b,1, Dolores Bussoc,1, Li Wanga,1, Dalia Galianid, Tony Chavarriae, Eliza Vasilef,
Linda Quilaqueoc, Juan Andrés Orellanag, Dalia Walzera, Ruth Shalgih, Nava Dekeld, David F. Albertinii, Attilio Rigottic,
David C. Pagea,b, and Monty Kriegera,2
Departments of aBiology and eComparative Medicine and fThe Koch Institute, Massachusetts Institute of Technology, Cambridge, MA 02139; bHoward
Hughes Medical Institute, Whitehead Institute, Cambridge, MA 02142; cDepartamento de Nutrición, Diabetes y Metabolismo and gDepartamento de
Neurología, Pontificia Universidad Católica de Chile, 8330024 Santiago, Chile; dDepartment of Biological Regulation, The Weizmann Institute of Science,
Rehovot 76100, Israel; hDepartment of Cell and Developmental Biology, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv 69978, Israel; and
i
Department of Molecular and Integrative Physiology, Institute for Reproductive Health and Regenerative Medicine, University of Kansas Medical Center,
Kansas City, KS 66160
Contributed by Monty Krieger, October 7, 2014 (sent for review July 22, 2013)
The HDL receptor scavenger receptor, class B type I (SR-BI) controls
the structure and fate of plasma HDL. Female SR-BI KO mice are
infertile, apparently because of their abnormal cholesterol-enriched
HDL particles. We examined the growth and meiotic progression of
SR-BI KO oocytes and found that they underwent normal germinal
vesicle breakdown; however, SR-BI KO eggs, which had accumulated excess cholesterol in vivo, spontaneously activated, and they
escaped metaphase II (MII) arrest and progressed to pronuclear, MIII,
and anaphase/telophase III stages. Eggs from fertile WT mice were
activated when loaded in vitro with excess cholesterol by a cholesterol/methyl-β-cyclodextrin complex, phenocopying SR-BI KO
oocytes. In vitro cholesterol loading of eggs induced reduction in
maturation promoting factor and MAPK activities, elevation of intracellular calcium, extrusion of a second polar body, and progression to meiotic stages beyond MII. These results suggest that the
infertility of SR-BI KO females is caused, at least in part, by excess
cholesterol in eggs inducing premature activation and that cholesterol can activate WT mouse eggs to escape from MII arrest. Analysis
of SR-BI KO female infertility raises the possibility that abnormalities
in cholesterol metabolism might underlie some cases of human female infertility of unknown etiology.
fertility
| egg | cholesterol | HDL | meiosis
S
cavenger receptor, class B type I (SR-BI) is an HDL receptor
that transports unesterified cholesterol (UC) and its esters
between lipoproteins and cells (1–3) and functions as a signaling
receptor (4). SR-BI controls the structure and composition of
plasma HDL and the amounts and fates of HDL cholesterol (2,
3, 5, 6). Homozygous null SR-BI knockout (KO) mice exhibit
hypercholesterolemia with unusually large and UC-enriched
HDL particles [abnormally high UC to total cholesterol (TC)
ratio] (5, 7, 8). This unusual hypercholesterolemia apparently
induces a deleterious accumulation of UC in RBCs and platelets
that influences their maturation, lifetime in the bloodstream,
structure, and/or function (7–13).
Female but not male SR-BI KO mice exhibit fully penetrant
infertility, despite their essentially normal ovarian histology (6,
14). Several lines of surgical transplantation, genetic, histologic,
and pharmacologic evidence indicate that the abnormal structure and composition of circulating HDL in SR-BI KO mice
contribute to their female infertility (Discussion). This infertility
can be effectively corrected by altering (through a variety of
approaches) the structure and abundance of the circulating HDL
to which the ovaries are exposed (7, 14, 15). This evidence also
suggests that the infertility is likely caused by defects in oocytes/
eggs manifested during the periovulatory period without affecting the primordial follicle pool. Indeed, ∼19% of the ovulated
eggs harvested ∼16 h after hormone-induced superovulation are
dead (6, 14) (see below). The remaining oocytes are unable to be
fertilized or develop into viable pups. Here, we examined SR-BI
E4972–E4980 | PNAS | Published online November 3, 2014
KO oocyte growth and progression through the peri- and postovulatory stages of meiosis (Fig. 1) and investigated the possibility
that high levels of UC in HDL might result in excess cholesterol
deposition in oocytes or eggs that might influence their growth,
meiotic progression, and/or viability.
Normally, after the luteinizing hormone surge of the estrous
cycle and just before ovulation, mammalian oocytes complete
the first meiotic division [meiosis I (MI)] and immediately
thereafter proceed to metaphase II (MII), a stage at which they
arrest (Fig. 1). A complex network of proteins and intracellular
signals establishes and maintains MII arrest [e.g., elevated
MAPK and maturation promoting factor (MPF; or cdk1/cyclin
B) kinase activities] (16, 17) and subsequently permits exit from
this stage after fertilization (e.g., reduction in MAPK and MPF
activities) (16, 17). Productive fertilization of MII-arrested eggs
leads to inositol triphosphate-induced oscillations in intracellular
calcium concentration ([Ca2+]i) that activate the eggs [exit from
MII arrest, second polar body (PB) extrusion, formation of
pronuclei, etc.] (Fig. 1) (18, 19). Fertilization-induced changes in
intracellular zinc levels also may influence activation (19). The
parthenogenetic stimulant SrCl2 activates normal eggs (20) by
inducing oscillations in [Ca2+]i (21, 22) and suppressing MAPK
and MPF kinase activities (23) that mimic those that occur after
fertilization (21, 22). Brief exposure to ethanol induces a single
spike in [Ca2+]i and activates eggs (24, 25). Unlike fertilized eggs,
Significance
Production of functional sperm and eggs requires a complex
process called meiosis. Meiosis in mouse and human eggs
pauses at a stage called metaphase II (MII) arrest until fertilization by sperm. After fertilization, eggs released from MII
arrest complete meiosis and develop into new individuals. In
analyzing the female infertility of genetically altered mice, we
discovered that excess cholesterol can trick mouse eggs into
behaving as though they were fertilized (released from arrest),
thus disrupting the normal synchrony between fertilization
and completion of meiosis and rendering them dysfunctional.
These findings suggest that abnormal cholesterol metabolism
may contribute to some forms of human female infertility.
Author contributions: A.Y., G.A.D., D.B., L.W., D.G., E.V., R.S., N.D., D.F.A., A.R., D.C.P., and
M.K. designed research; A.Y., G.A.D., D.B., L.W., T.C., E.V., L.Q., and D.W. performed
research; J.A.O. contributed new reagents/analytic tools; A.Y., G.A.D., D.B., L.W., E.V.,
D.C.P., and M.K. analyzed data; and A.Y., G.A.D., D.B., L.W., R.S., N.D., and M.K. wrote
the paper.
The authors declare no conflict of interest.
1
A.Y., G.A.D., D.B., and L.W. contributed equally to this work.
2
To whom correspondence should be addressed. Email: krieger@mit.edu.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1418954111/-/DCSupplemental.
www.pnas.org/cgi/doi/10.1073/pnas.1418954111
PNAS PLUS
which receive the paternal chromosome complement from the
sperm, SrCl2- or ethanol-activated eggs are haploid (Fig. 1) and
cannot support proper embryogenesis (22).
Here, we found that, within follicles of hormone-treated SRBI KO females, oocytes apparently resumed meiosis normally.
However, these cells spontaneously escaped MII arrest, exhibited
reduced MPF and MAPK activities, and aberrantly progressed
to haploid pronuclear, metaphase III, and anaphase/telophase III
stages, similar to normal ovulated eggs after chemical (e.g., SrCl2)
(22, 25) activation (Fig. 1). Compared with control eggs, the eggs
of SR-BI KO females accumulated excess cholesterol. Strikingly,
we could recapitulate the apparently spontaneous activation of
ovulated SR-BI KO eggs in eggs ovulated from fertile control
mice (WT or SR-BI+/−) by treating those eggs with a cholesterol/
methyl-β-cyclodextrin (MβCD) complex to load them with excess
cholesterol (26). In addition, cholesterol loading induced a single
spike in [Ca2+]i reminiscent of that seen in ethanol-activated eggs
(24, 25). These results establish that excess cholesterol loading
provides a new approach to activating mammalian MII-arrested
eggs and suggest that the infertility of SR-BI KO females is
caused, at least in part, by excess cholesterol acting directly on the
egg to induce premature activation. Analysis of SR-BI KO female
infertility also raises the possibility that abnormalities in cholesterol metabolism might underlie some forms of human female
infertility of unknown etiology.
Yesilaltay et al.
Results
Our previous studies involving restoration of fertility by ovary
transplantation and drug treatment suggested that the infertility
of SR-BI KO (SR-BI−/−) female mice is unlikely to be caused
by irreversibly impaired early oogenesis or abnormal folliculogenesis (2, 6, 7, 14) (Discussion). Rather, the infertility seems
likely to be related to defects in oocyte growth, maturation, or
activation. SR-BI KO females ovulate almost normal numbers
of eggs (6). However, on their recovery from the oviduct, a substantial fraction of the ovulated eggs displays an abnormal,
nonrefractile morphology characteristic of dead eggs (the criterion used to score for death here). On average, 4% of ovulated
eggs from control SR-BI+/− females (n = 47 animals) and 19% of
eggs from SR-BI KO females (n = 39 animals) were dead when
harvested at 13–16 h after induction of superovulation with human chorionic gonadotropin (hCG) (Materials and Methods).
The death rate of eggs from SR-BI KO females increased to 53%
(n = 7) when they were collected 18–19 h post-hCG injection.
Moreover, the viable ovulated eggs cannot be successfully fertilized in vivo to form either early embryos or viable pups. In an
effort to better understand the infertility of SR-BI KO females,
we investigated events in oocyte development after the SR-BI−/−
oocytes are recruited from the primordial follicle pool. For many
of the experiments reported here, we compared results obtained
from SR-BI KO oocytes with those from control SR-BI+/− and
WT SR-BI+/+ oocytes or eggs, all of which are from mice on
PNAS | Published online November 3, 2014 | E4973
MEDICAL SCIENCES
Fig. 1. Meiotic resumption and egg activation in the mouse. Schematic representation of meiotic resumption and egg activation by either fertilization or
chemical agents. The stages at which primary (prophase I) and secondary (MII) arrest occur are indicated. Oocytes initiate meiosis during fetal development
and enter primary arrest at the diplotene stage of prophase I around the time of birth (not illustrated here). Prophase I arrest is maintained during oocyte
growth and differentiation. Prior to ovulation, meiosis resumes, the nuclear envelope of the germinal vesicle (GV) dissassembles [GV breakdown (GVBD)], and
chromosomes condense and align on the first metaphase plate. During the first division, homologous chromosomes segregate and the first polar body (PB) is
extruded (first PB in anaphase/telophase I). The remaining chromosomes realign on the second metaphase plate (MII) followed by secondary arrest. During
MII arrest, the first PB often degrades. Eggs that are ovulated can exit MII arrest and complete meiosis when activated by a fertilizing sperm (upper right) or
chemical agents, such as SrCl2 or ethanol (lower right), which induce a spike (ethanol) or oscillations (sperm and SrCl2) in cytoplasmic Ca2+ levels and suppress
MAPK and MPF activities. On activation, sister chromatids segregate, and the second PB is extruded (second PB in anaphase/telophase II). The activated egg
then progresses to pronuclear stage as the nuclear envelope reforms. On chemical activation, the egg can alternatively progress to metaphase III, segregate
the remaining chromatids randomly during anaphase III, and extrude a third PB (anaphase/telophase III).
a 50:50 C57BL/6:129 genetic background (Materials and Methods). In some experiments, we used eggs from females on a WT,
pure C57BL/6 background.
Oocyte Follicular Growth. Oocytes of the primordial follicle pool
arrested in prophase I of meiosis (Fig. 1) are recruited to grow
and differentiate throughout the mouse’s reproductive lifetime.
As oocytes grow, their volume increases proportionally to the
volume of the encasing follicle. In rodents, the relative oocyte to
follicle diameter from intact follicles of unprimed females has
been used as an indicator of follicular oocyte growth (27, 28). We
mechanically isolated murine follicles from both SR-BI KO mice
and fertile SR-BI+/− controls at different stages of development
from unprimed sexually immature (prepubertal) 3-wk-old and
mature 6-wk-old females. The oocyte and follicle diameters were
measured as described in Materials and Methods. Fig. 2 A and B
shows that, at both ages, there were linear relationships between
the oocyte and follicle diameters and that the slopes of these
lines were virtually identical for the follicles and oocytes from
SR-BI KO females and SR-BI+/− controls. Thus, SR-BI deficiency does not seem to alter the growth of oocytes and their
surrounding follicles.
Meiotic Resumption and Extrusion of the First PB. An important
indicator of oocyte quality is the ability to resume and complete
meiosis, a process called maturation (29). In mice, oocytes initiate meiosis during fetal development and progress through
Fig. 2. Oocyte growth and meiotic resumption. (A and B) Follicles were
mechanically isolated from either fertile control SR-BI+/− (black circles and
thick lines) or infertile SR-BI KO (SR-BI−/−; white circles and thin lines) mice at
(A) 3 (unprimed sexually immature) or (B) 6 wk of age (mature). The follicles
were fixed, and follicle and oocyte diameters were measured as described in
SI Materials and Methods. The almost superimposed thick and thin lines
represent results of linear least squares analysis. The numbers of follicles/
oocytes examined at 3 and 6 wk were control SR-BI+/− (49 and 89, respectively) and SR-BI KO (50 and 107, respectively). (C) GVBD is a spontaneous and dynamic process that begins as early as 30 min after oocytes are
placed in culture and reaches its maximum 4–6 h later. Two- to five-mo-old
control SR-BI+/+ (black circles) and SR-BI KO (white squares) females were
synchronized by priming with pregnant mare serum gonadotropin; 46 h
later, GV oocytes were retrieved by follicular puncture and cultured in M16
media containing the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) to maintain high levels of cAMP and thus, arrest at the GV
stage. The cumulus cells were removed (SI Materials and Methods), and the
cumulus-free oocytes were transferred to M16 medium without IBMX. At
the indicated times thereafter, the percentage of oocytes undergoing GVBD
was scored by microscopic examination. The data were pooled from two
independent experiments with ≥10 oocytes per female and three females of
each genotype per experiment.
E4974 | www.pnas.org/cgi/doi/10.1073/pnas.1418954111
prophase I until the diplotene stage, where they enter primary
arrest (Fig. 1). These primordial oocytes acquire the competence
to resume meiosis during the late stages of growth and differentiation. In full-grown oocytes, primary arrest is maintained by
the follicular soma and relieved before ovulation. Oocytes that
resume meiosis undergo germinal vesicle breakdown (GVBD),
assemble chromosomes on the first metaphase plate, and complete the first meiotic division, which is accompanied by extrusion of the first PB (Fig. 1). Oocytes then assemble the remaining
single set of homologous chromosomes on the second metaphase
plate and enter secondary arrest (MII arrest) (Fig. 1).
Full-grown, prophase I-arrested oocytes in the ovary can be
triggered to resume meiosis and progress to MII and secondary
arrest by removing them from their follicles. Thus, to investigate
whether full-grown oocytes from the SR-BI KO mice and SRBI+/− controls were competent to resume meiosis, we isolated
them from ovaries of unprimed females by follicular puncture
and cultured them for 14 h. Resumption and completion of MI
were scored by determining by microscopic examination GVBD
and first PB extrusion, respectively (Materials and Methods).
Table S1 shows that the fractions of SR-BI+/− and SR-BI KO
oocytes undergoing GVBD were similar (87% and 74%, respectively), and the fractions of oocytes extruding the first PB
were also similar (78% and 64%, respectively). Fig. 2C shows
that the rate of GVBD in oocytes from SR-BI KO animals was
similar to that for SR-BI+/− controls. These results indicate that
the rate at and extent to which SR-BI KO oocytes resume
(GVBD) and complete MI (first PB extrusion) are similar to
those of control, fertile oocytes, indicating that they are competent to resume meiosis.
Spontaneous Progression Beyond MII Arrest. After completion of
MI, WT eggs, which still reside in the ovarian follicles, arrest at
MII, at which point they are ovulated. The ovulated eggs in the
oviduct remain arrested at MII until they are activated by sperm
during fertilization, which triggers the completion of meiosis II
and the extrusion of the second PB (Fig. 1). Eggs can also activate spontaneously (e.g., during aging) or artificially (e.g., by
treatment with chemicals, such as SrCl2/EGTA, ionophores, or
ethanol) (30–33). To study postovulatory events in eggs from SRBI KO mice, we harvested morphologically live eggs from the
oviducts of hormone-primed SR-BI KO and control SR-BI+/−
females either ∼16 or ∼20 h after hCG administration. We determined the meiotic stage of both control SR-BI+/− and SR-BI
KO eggs by examining their chromosome/nuclear and spindle
organizations. Fig. 3 shows eggs that were stained using a wholemount preparation technique and imaged by deconvolution microscopy to visualize the DNA (DAPI, blue), microtubules (antitubulin, green), nuclear envelope (anti-lamin B1, see Fig. S1), and
centromeres (anti-centromere antibody, red). We present the 3D
organization of chromosomes in the egg and its PB as a z stack of
deconvolved images. After image collection and processing, we
scored the meiotic state of each egg by determining the number of
chromatids by counting centromeres (one chromatid per centromere) in the egg and its PB as well as scoring the morphology of
the spindle and the presence or absence of the nuclear lamina. The
results are presented in Table 1, and representative images are
shown in Fig. 3.
The two leftmost columns in Fig. 3 show control SR-BI+/− and
SR-BI KO eggs at metaphase II contained 40 sister chromatids
(20 pairs) aligned on a spindle (Fig. 3, two leftmost columns,
spindles perpendicular to the plane of the image). The other SRBI KO eggs were classified, based on their chromosomal and
spindle morphologies, into five categories, for which in Fig. 3
schematic representations (top row) and representative images
at several magnifications (rightmost five columns) are shown. In
each case, progression beyond MII was evident from the segregation of sister chromatids (40 at MII), leaving 20 single chromatids
Yesilaltay et al.
PNAS PLUS
within the egg and as expected, 20 single centromeres per second
PB (Fig. 3 and Fig. S2). Formation of a pronucleus was confirmed
by staining for lamin B1, a component of the nuclear lamina that
would be indicative of exit from meiosis and entry into the first
mitotic cell cycle (Fig. S1).
Most of the control SR-BI+/− eggs (n = 8) were either arrested
at MII (Fig. 3, first column) or at an earlier stage at ∼16 (93% of
eggs scored) or ∼20 h (89%) after hCG administration (induction
of superovulation) (Table 1). In this experiment, a small fraction of
the control SR-BI+/− eggs had progressed beyond MII. In addition,
no WT SR-BI+/+ eggs progressed beyond MII at ∼16 h (n = 8),
indicating that the control SR-BI+/− eggs may exhibit a mild defect
Table 1. Quantitative morphologic analysis of spontaneous
meiotic progression beyond MII arrest
Hours after hCG administration
∼16
Stage in meiotic
progression
At MII or earlier (%)
Beyond MII (%)
+/−
∼20
+/−
SR-BI
(n = 30)
SR-BI KO
(n = 11)*
SR-BI
(n = 9)
SR-BI KO
(n = 27)
93
7
27
73
89
11
7
93
*Four SR-BI KO oocytes at ∼16 h and two SR-BI KO oocytes at ∼20 h could
not be scored, because the chromatin was too diffuse to reliably count
centromeres.
Yesilaltay et al.
in MII arrest. In other experiments, we observed no differences in
the very low level of spontaneous activation between WT and heterozygous mutant eggs. Table 1 shows a dramatic time-dependent
increase in SR-BI KO eggs that had progressed past MII at ∼16
(73%) or ∼20 h (93%) (representative images are in Fig. 3).
Activation of ovulated eggs by either fertilization or parthenogenetic agents suppresses the kinase activities of MPF and
MAPK that are required to maintain MII arrest (16, 17, 23). We
determined the MPF and MAPK activities of lysates from ovulated eggs that were harvested 18 h after hCG administration,
incubated for 6 h in M16 medium, and then, collected in pools of
four eggs each: SR-BI+/+, five pools; SR-BI+/−, seven pools; SRBI−/− (KO), nine pools (additional details in SI Materials and
Methods). Few of the eggs from the SR-BI+/+ (5 of 39) and SRBI+/− (1 of 31) mice exhibited second PBs, whereas eggs from the
SR-BI KO either did (all eggs in six pools) or did not (no eggs in
three pools) exhibit second PBs. Fig. S3 shows that there were
substantially lower MPF and MAPK activities in SR-BI KO eggs
that spontaneously escaped MII arrest (e.g., second PB extrusion) compared with those without second PBs from both the
control and the SR-BI KO mice. Thus, at least some of the
biochemical processes that normally accompany egg activation
were spontaneously induced in the ovulated SR-BI KO eggs that
were activated.
Because most SR-BI KO eggs failed to arrest at MII, which
was indicated by progression to anaphase/telophase II, completed meiosis II, pronuclear stage, metaphase III, or anaphase/
telophase III (Fig. 1), and exhibited reduced MPF and MAPK
PNAS | Published online November 3, 2014 | E4975
MEDICAL SCIENCES
Fig. 3. Meiotic progression of oocytes from control SR-BI+/− and SR-BI KO mice assessed by deconvolution microscopy. SR-BI+/− and SR-BI KO females were
superovulated, and oocytes were harvested ∼20 h after hCG administration and denuded by hyaluronidase treatment. The oocytes were stained to visualize
chromosomes (DAPI; blue), microtubules (tubulin; green), centromeres [anticentromere antibody (ACA); red], and the nuclear membrane (lamin B1) (Fig. S1).
Deconvolved and z-projected images were collected. Representative images of oocytes at various stages of meiosis (schematically indicated in the top row) are
shown. For clarity, only 8 of 40 chromatids present at MII arrest are illustrated in the top row. Low magnification images (second row) show the overall
morphology of the eggs; arrows indicate second polar bodies. Medium magnification images (third row) show the arrangement of the chromosomes and the
spindle; dashed lines outline the cells and second polar bodies. High magnification images (bottom row) illustrate the numbers and arrangements of
chromatids (red spots) in the eggs. High magnification images of the chromatids in representative polar bodies may be found in Fig. S2. Scale bars (10 μm) for
each row are shown in the leftmost images, except that the magnification of the “high mag.” image for anaphase/telophase III (lower rightmost panel) differs
slightly from those of the other high magnification images as indicated by its own scale bar (10 μm).
Fig. 4. Cholesterol levels in SR-BI+/+ and SR-BI KO eggs. Eggs harvested ∼16 h
after hCG administration from superovulated (A) SR-BI+/+ and (B) SR-BI KO
mice on a mixed C57BL/6:129 background were denuded by hyaluronidase
treatment, washed, incubated with 0.5 mg/mL Filipin III complex (Sigma),
washed again, mounted on slides, and imaged by fluorescence microscopy
(UV light excitation) as described in SI Materials and Methods. The images
shown are representative of those observed in two independent experiments using at least six eggs for each genotype. (Scale bars: 20 μm.)
activities, impairment of cell cycle control may, in part, account
for SR-BI KO female infertility, although other factors may
contribute as well.
Abnormally High Cholesterol Deposition in SR-BI KO Eggs. In SR-BI
KO mice, high cholesterol deposition in RBCs and platelets,
caused at least in part by the high UC content of HDL, contributes to diverse abnormalities of these cells (5, 9, 10, 12, 13).
We suspected that SR-BI KO eggs might have abnormally high
cholesterol accumulation that might contribute to the spontaneous escape of ovulated SR-BI KO eggs from MII arrest. Thus,
we measured the relative UC levels in ovulated eggs from hormone-primed mice by fluorescence microscopic analysis of cells
stained with the cholesterol-binding fluorescent dye filipin (34).
Fig. 4A shows that filipin fluorescence in control eggs from
SR-BI+/+ females exhibited a weak, uniform intensity together with
somewhat brighter puncta. Similar results were observed using
eggs from SR-BI+/− females. Eggs from SR-BI KO females (Fig.
4B) showed a similar staining pattern, except that the intensity
of fluorescence was much stronger (68% ± 15% increase, P =
0.004) (Fig. S4). These results indicate the presence of abnormally high levels of UC in the SR-BI KO eggs and raise the
possibility that excess UC in SR-BI KO eggs might be responsible for their abnormal escape from MII arrest and possibly
might contribute to premature death. Loading cells with excess
UC by various routes can induce endoplasmic reticulum (ER)
stress, mitochondrial dysfunction, and mitochondrial-mediated
cell death (35–41).
Effects of Excess Cholesterol Loading on WT Eggs. We directly
assessed the ability to activate eggs from WT mice by loading them
with excess cholesterol. Exogenous cholesterol was delivered by
treating eggs for 10–45 min with 0.125 or 0.5 mM cholesterolloaded MβCD (+ cholesterol) as indicated, a standard approach
for increasing cellular cholesterol content (26, 42). For example,
incubating SR-BI+/+ eggs for 10 min at 37 °C with 0.5 mM cholesterol/MβCD increased relative UC by 38% ± 10% (P = 0.019)
(Fig. S4). Eggs isolated from the oviducts of hormone-primed WT
C57BL/6 females ∼14–16 h after hCG administration were incubated with or without cholesterol/MβCD, washed to remove
the cholesterol/MβCD, and then, incubated without cholesterol/
MβCD for an additional 5–6 h at 37 °C. For comparison, some eggs
were incubated for 6 h with the well-established chemical activator
5 mM SrCl2 in calcium-free or 2 mM EGTA-containing medium.
We examined several phenotypes associated with egg activation, including (i) alterations in [Ca2+]i measured immediately
after cholesterol/MβCD addition (assessed using the Ca2+-senE4976 | www.pnas.org/cgi/doi/10.1073/pnas.1418954111
sitive dye Fura-2AM), (ii) MPF and MAPK activities, (iii)
second PB extrusion, (iv) stage in meiosis (deconvolution
microscopy), and (v) subsequent cleavage to the two-cell stage.
Fig. 5 shows typical time courses of the changes in the relative
[Ca2+]i after cholesterol addition for five individual WT C57BL/6
eggs continuously incubated with cholesterol/MβCD for 45 min.
For all of the eggs that remained viable during the experiments
(n = 26), the exposure to cholesterol/MβCD induced a single
relatively steep increase in [Ca2+]i after an ∼15- to 34-min lag.
For most of these eggs (24 of 26), the increase was transient,
with a partial (3 of 24) or essentially complete (21 of 24) (Fig. 5)
return to the baseline before the end of the 45-min imaging
period. There was variation in the amplitude and width of the
spikes in different eggs. The single spike in [Ca2+]i was somewhat
reminiscent of that seen when transient ethanol exposure induces activation (24, 25) and differed from the robust oscillations
observed by SrCl2 or fertilization-induced activation (18, 19, 21,
22, 43) (Fig. S5). We also performed [Ca2+]i imaging experiments on eggs exposed to cholesterol/MβCD for 25 min, in which
we imaged the eggs during cholesterol/MβCD exposure and
then, after the cholesterol/MβCD was washed away. We observed a single large spike in [Ca2+]i that returned to baseline
when we collected data between 20 and 155 min after removing
the cholesterol/MβCD (Fig. S6). There was no evidence of [Ca2+]i
oscillations induced by cholesterol/MβCD.
Fig. 6 shows that cholesterol loading for 20–25 min seemed to
be as effective as SrCl2 treatment in activating the eggs, which
was assessed by suppression of MAPK and MPF kinase activities
(Fig. 6A), induction of PB extrusion (Fig. 6 B and C), and progression to subsequent stages in meiosis (e.g., pronucleus formation) (Fig. 6C). All three assessments were made ∼5–6 h after
the addition of cholesterol or SrCl2. Comparable cholesterol
induction of second PB extrusion was observed using eggs from
hormone-primed SR-BI+/− mice on a mixed C57BL/6:129 background (Fig. S7). We also measured second PB extrusion in WT
C57BL/6 eggs during [Ca2+]i imaging experiments, in which we
imaged the eggs during the 25-min cholesterol/MβCD exposure
and for an additional 155 min after the cholesterol/MβCD was
washed away. A single [Ca2+]i spike was observed in most of the
Fig. 5. Effects of cholesterol loading on [Ca2+]i in eggs from WT C57BL/6
females. Eggs from WT C57BL/6 females were harvested ∼14–16 h after hCG
administration, stripped of cumulus cells using hyaluronidase, and loaded
with the calcium-sensitive fluorescent dye Fura-2AM. The eggs were then
placed at time = 0 in M2 medium supplemented with 0.125 mM cholesterol/
MβCD (Chol/MβCD) at 37 °C. Relative [Ca2+]i measured using an inverted
microscope as a function of time (sampled every 10 s) was determined as the
ratio of fluorescence intensities excited by 340- and 380-nm light. Eggs in
Chol/MβCD-containing medium were continuously imaged, and the results
for five typical eggs are shown displaced from each other on the y axis by 2
arbitrary units to prevent overlap. After 45 min of Chol/MβCD exposure, the
eggs were washed and placed in Chol/MβCD-free M2 medium at 37 °C for an
additional ∼5.25 h, after which second PB extrusion was assessed.
Yesilaltay et al.
Discussion
Female SR-BI KO mice exhibit fully penetrant infertility (6, 14).
Our previous findings that these animals have essentially normal
ovarian histology and that their fertility can be readily rescued by
an HDL cholesterol-lowering drug suggested that the infertility
might be a consequence of defects in oocytes or eggs, possibly
caused by the animals’ unusual dyslipidemia (hypercholesterolemia with abnormal HDL structure and composition) (2, 6, 7, 14,
15). Indeed, defects occurring during or soon after ovulation in
vivo are readily apparent, because ∼19% of the eggs harvested
∼16 h after hCG administration (hormone-induced superovulation) are dead (refs. 6 and 14 and this study).
Here, we sought to understand the cellular basis of SR-BI KO
female infertility by investigating oocyte growth and meiotic
progression. We observed no obvious defects in oocyte growth in
the follicle, maintenance of primary meiotic arrest in prophase I,
resumption of meiosis after release from primary arrest (kinetics
of GVBD), or efficiency of first PB extrusion (Fig. 1 shows a
schematic depiction of meiosis and parthenogenesis). However,
when we examined ovulated eggs from control and SR-BI KO
females, we found that substantial numbers of SR-BI KO eggs
had spontaneously activated (escaped MII arrest and exhibited
second PB extrusion and suppression of MFP and MAPK activities). Deconvolution immunofluorescence imaging of the
untreated, viable SR-BI KO eggs harvested at ∼16 or ∼20 h after
hCG administration established that the progressive spontaneous escape from MII arrest resulted in second PB extrusion,
pronucleus formation, and progression to metaphase III and
Fig. 6. Additional effects of cholesterol loading and SrCl2 treatment on eggs from WT C57BL/6 females. (A–C) Eggs from WT C57BL/6 females were harvested
∼14–16 h after hCG administration and stripped of cumulus cells by hyaluronidase treatment. (A) MAPK and MPF kinase activities. Eggs were pretreated for
25 min without (−) or with (+) 0.125 mM cholesterol-loaded MβCD (Cholesterol) in M16 at 37 °C as indicated. They were then washed and incubated in M16
medium either without [for untreated (n = 60) or cholesterol-loaded MβCD (n = 68) eggs] or with 5 mM SrCl2/2 mM EGTA (n = 40) at 37 °C for 6 h. Eggs were
then washed, pooled into sets of four, and lysed, and MAPK and MPF kinase activities were measured as described in SI Materials and Methods. The values
represent the averages ± SEMs from four independent assays using eggs harvested and processed on 7 different d. The values for one-way ANOVA for MPF
and MAPK activities were P < 0.00001. *P value < 0.02 (unpaired Student t test) for comparisons with SrCl2; †P value < 0.0001 (unpaired Student t test) for
comparisons with untreated controls; #P value < 0.001(unpaired Student t test) for comparisons with SrCl2. (B) PB extrusion. Some of the eggs (n = 124) were
pretreated at 37 °C for 25 min with (+) 0.125 mM cholesterol-loaded MβCD (Cholesterol) in M16 medium and then washed and incubated in M16 medium for
an additional 6 h at 37 °C. The remaining eggs were incubated for 6 h at 37 °C in M16 medium without (n = 103) or with (n = 114) 5 mM SrCl2/2 mM EGTA. The
fraction of eggs exhibiting second PB extrusion was then determined (averages from eight independent experiments). The value for one-way ANOVA was P <
0.00001. †P value < 0.0001 (paired Student t test) for comparisons with untreated controls. (C) Deconvolution microscopy. Eggs were pretreated for 20 min
without (−) or with (+) 0.5 mM cholesterol-loaded MβCD (Cholesterol) in M16 at 37 °C as indicated. They were then washed and incubated in M16 medium
either without [for untreated (n = 20) or cholesterol-loaded (n = 20) eggs] or with 5 mM SrCl2/2 mM EGTA (n = 17) at 37 °C for 6 h. We then stained the cells
and collected deconvolved and z-projected images as described in Fig. 3. (Upper) Low-magnification images show the overall morphology of the eggs; dashed
lines outline the cells and the second PBs. (Lower) High-magnification images show the number and arrangement of chromatids in either the main cell
[untreated; 40 paired centromeres (red) at the MII spindle] or the second PBs (SrCl2 and cholesterol). The fractions of eggs that extruded a second PB were
untreated, 0% (0 of 18); SrCl2, 77% (10 of 13); and cholesterol/MβCD, 74% (14 of 19). Those eggs that were fragmented at the end of the 6-h incubation
(untreated, two; SrCl2, four; cholesterol, one) were not included in the calculation of the percentage exhibiting second PB exclusion. ACA, anticentromere
antibody. (Scale bar: 10 μm.)
Yesilaltay et al.
PNAS PLUS
terol delivery can induce activation, suggesting that spontaneous
activation caused by excess cholesterol in eggs of SR-BI KO females
contributes directly to their female infertility.
PNAS | Published online November 3, 2014 | E4977
MEDICAL SCIENCES
viable eggs (16 of 18 in three independent experiments) (Fig.
S6B), and 94% (15 of 16) of those eggs extruded a second PB by
the end of the second 155-min cholesterol/MβCD-free incubation.
Thus, multiple distinct [Ca2+]i spikes were not required for cholesterol-induced second PB extrusion.
In multiple experiments, we found that most cholesterol/
MβCD-activated eggs progressed to the two-cell stage 24 h after
treatment. For example, in two such experiments, we followed
for 24 h in vitro the fates of 0.125 (n = 41) or 0.5 mM (n = 11)
cholesterol-loaded WT C57BL/6 eggs; of those eggs that extruded second PBs after 6 h (68% and 73%, respectively), 71%
and 63%, respectively, underwent cleavage to the two-cell stage
by 24 h after cholesterol loading. In contrast, untreated eggs
remained arrested in MII. Strikingly, in our [Ca2+]i imaging
experiments on C57BL/6 eggs loaded with cholesterol for
25 min, of those eggs that were viable 24 h after treatment (n = 29),
90% extruded a second PB at ∼6 h (n = 26), 100% of which
exhibited a Ca2+ spike. In addition, 86% of those activated eggs
that were examined (19 of 22) were found to have progressed to
the two-cell stage. Of the remaining three eggs that had not
extruded a second PB, two eggs exhibited a Ca2+ spike, and none
progressed to the two-cell stage (additional details in SI Materials
and Methods). Thus, induction of a Ca2+ spike may be necessary
but not sufficient for cholesterol/MβCD-induced egg activation.
Future experiments will be required to determine which features of the Ca2+ spike (onset time, duration, amplitude, etc.)
and the status of the eggs (e.g., extent of cholesterol loading) are
necessary for activation. Taken together, our observations indicate
that delivery of exogenous cholesterol to eggs efficiently induces
exit from MII arrest by mechanisms that include biochemical
changes associated with classic chemical-mediated activation
and sperm-mediated activation during fertilization. Thus, choles-
anaphase/telophase III. Spontaneous fertilization-independent
exit from MII (or inability to establish or maintain arrest at MII)
results in cells with a haploid chromosome complement, which
in at least some cases, is further reduced during subsequent
progression to metaphase III and anaphase/telophase III. These
cellular defects are likely to account for at least some and perhaps most of the inability of ovulated SR-BI eggs to be fertilized
and develop normally into viable embryos after mating (6).
Additional studies are necessary to determine whether the SR-BI
KO eggs that were dead at harvesting ∼16 h posthormone induction died because of meiotic defects or if other independent
abnormalities also contributed to this process.
Cholesterol Accumulation in SR-BI KO Eggs Contributes to Their
Abnormal Phenotypes. One potential mechanism underlying pre-
mature death and spontaneous escape from MII arrest of SR-BI
KO eggs is their abnormal accumulation of cholesterol. Indeed,
excess cholesterol accumulation in RBCs and platelets in SR-BI
KO mice, apparently caused by the high UC:TC ratio in circulating HDL (7, 8), has previously been associated with the abnormal maturation, lifetime, structure, and/or function of these
blood cells (7, 9–13). As was the case for RBCs and platelets in
SR-BI KO mice, we detected excess cholesterol in ovulated SRBI KO eggs (here assessed by filipin staining). In complementary
experiments, we found that cholesterol loading of control, fertile,
SR-BI+/−, or WT eggs by incubation with a cholesterol/MβCD
complex resulted in activation similar to that of SR-BI KO eggs
as well as a single spike in [Ca2+]i. Prolonged accumulation of
excess cholesterol might also contribute through various routes
(e.g., ER stress, mitochondrial dysfunction, and excessive or
prolonged [Ca2+]i elevation) to the premature death of some of
the SR-BI KO eggs (35–41, 44, 45). We, therefore, conclude that
(i) cholesterol can induce parthenogenetic activation of mouse
eggs and that (ii) accumulation of excess cholesterol in SR-BI
KO oocytes plays a substantial role in generating their abnormal
phenotypes and consequently, the infertility of SR-BI KO females.
Conversely, Buschiazzo et al. (46) have shown that cholesterol
depletion by exposure to high levels of cholesterol-free MβCD
(15 mM for 30 min at 37 °C) induces a delay in second PB extrusion and a low fertilization of mouse eggs. We have found that
pretreating SR-BI+/− and SR-BI KO eggs with 10 mM cholesterolfree MβCD reduces filipin staining and is severely toxic (81% and
88% reduced viability, respectively). Taken together, these data
suggest that cholesterol in mouse eggs must be maintained within
a relatively narrow physiological range to ensure the oocyte’s health
and developmental potential. A critical role of UC in mediating
oocyte/egg abnormalities is consistent with our previous report that
correcting the abnormal composition (high UC:TC ratio) of plasma
HDL can restore fertility (7, 10, 14). It is possible that excess
cholesterol-independent mechanisms may also be involved.
HDL Is the Likely Source of Excess Cholesterol in SR-BI KO Eggs. Four
lines of surgical transplantation, genetic, and pharmacologic
evidence support the hypothesis that abnormal circulating HDL
in SR-BI KO mice (high UC:TC ratio and large diameter) (5, 7,
8) is a major cause of female SR-BI KO infertility and likely to
be the cause of excess cholesterol accumulation in their eggs. (i)
Ovary transplantation established that ovarian expression of SRBI is not necessary for fertility during embryonic development
(primordial follicle production) or adult maturation and function
(primordial follicle conversion to antral follicles and ovulation).
SR-BI KO ovaries bilaterally transplanted into SR-BI–positive
hosts developed the capacity to ovulate functional eggs that can
be fertilized and generate healthy pups (14). (ii) Tissue-specific
transgenic expression of SR-BI in the liver of SR-BI KO mice
restores not only normal HDL composition and structure but
also, essentially normal fertility (15). (iii) In SR-BI KO mice,
homozygous genetic ablation of the gene encoding apoA-I, the
E4978 | www.pnas.org/cgi/doi/10.1073/pnas.1418954111
major protein component of HDL, reduces total plasma cholesterol (mainly HDL cholesterol) to essentially WT levels, although the HDL particles remain abnormally large. Fertility is
partially restored in these double KO mice (41% fertility and
small litter sizes) (14). (iv) Oral administration of the HDL
cholesterol-lowering drug probucol reduces total plasma cholesterol to WT levels and restores to essentially normal the UC
content of HDL, although the HDL particles remain abnormally
large (7, 14). Fertility of SR-BI KO females is restored soon after
probucol treatment (14). Several studies indicate that the abnormal UC composition (high UC:TC ratio) rather than the increased
size of HDL particles in SR-BI KO mice may play a particularly
important role in SR-BI KO female infertility. As noted above,
apoA-I deficiency and probucol treatment partially or nearly fully
restore fertility to SR-BI KO mice without substantially reducing
the abnormally large size of HDL particles. Also, transgenic expression of the human cholesterol ester transfer protein in SR-BI
KO mice reduces the size/density of the HDL in a dose-dependent
fashion (47, 48). Although the size of the HDL particles in these
transgenic SR-BI KO mice is normalized, the composition is not
(abnormally high UC:TC ratio), and they are infertile (48).
There are two attractive mechanisms by which abnormal HDL
particles in the follicular fluid of antral follicles in SR-BI KO
mice might lead to excess cholesterol accumulation in oocytes.
Because of the relatively small size of HDL particles compared
with other lipoproteins, HDL in many animals is the only lipoprotein present in substantial amounts in the follicular fluid that
bathes the developing oocyte in the antral follicle (49–52). It may
be that, in the follicle, the UC-enriched HDL particles transfer
cholesterol to the oocytes directly, which they seem to do in RBCs
and platelets (9, 10), or indirectly (e.g., through surrounding cumulus cells). Su et al. (53) have suggested that mouse preovulatory
oocytes synthesize little cholesterol and obtain cholesterol primarily from the surrounding cumulus cells. Thus, there seems to
be a mechanism that could transfer HDL-derived cholesterol from
cumulus cells to the oocytes. An alternative mechanism for excess
cholesterol accumulation in SR-BI KO oocytes is that normal
cholesterol homeostasis in oocytes may depend on adequate levels
of HDL-mediated cholesterol efflux to balance input and that the
UC-enriched HDL particles in SR-BI KO mice may be unable to
mediate sufficient cholesterol efflux to prevent excess cholesterol
accumulation.
HDL has been shown to have multiple activities, including the
ability to transport lipids other than cholesterol [e.g., vitamin E
(54) and sphingosine-1-phosphate (55)] and influence oxidation,
inflammation, metabolism, chemotaxis, thrombosis, microbial
infection, cell signaling, and others (56–58). Thus, it is possible
that, in addition to contributing to accumulation of excess cholesterol in oocytes, some of the effects of the abnormal HDL on
female fertility in SR-BI KO mice may be independent of its role
in cholesterol transport.
Excess Cholesterol and Egg Activation. One possible mechanism by
which excess cholesterol might activate eggs is its stimulation of
an increase in cytoplasmic [Ca2+], possibly by inducing ER Ca2+
release through a stress pathway or by otherwise influencing
calcium channel or pump activities. Normally, fertilization or
parthenogenetic agents (e.g., SrCl2/EGTA or ethanol) induce a
single spike or oscillations in [Ca2+]i that are important for activation and successful progression of embryonic development
(18, 19, 24, 25, 43).
Excess cholesterol-induced escape from MII arrest is unlikely
to involve all of the same mechanisms as those induced by the
MEK inhibitor U0126 (blocks MAPK activation) (23, 59, 60),
c-mos deficiency (23, 61), or Zn depletion (25, 62, 63) because of
their differing phenotypes. Both c-mos KO and Zn-insufficient
eggs share with SR-BI eggs an otherwise spontaneous exit from
MII arrest (64). However, unlike SR-BI KO eggs, c-mos KO and
Yesilaltay et al.
Implications for Human Infertility. This study has uncovered a previously unidentified ability of excess cholesterol to activate MIIarrested mouse eggs. In addition, this report raises the possibility
that abnormalities in cholesterol metabolism in general and SRBI–mediated cholesterol metabolism specifically (6, 7, 14) have
the potential to contribute to human female infertility of unknown
etiology (72, 73). In such cases, pharmacologic manipulation of
cholesterol metabolism (e.g., lowering excess cholesterol in oocytes
or eggs) might prove beneficial. It is noteworthy that Velasco et al.
(74) and Yates et al. (75) have reported a potential influence of
variations in SR-BI on fertility of women undergoing in vitro
fertilization (IVF). In one study, Velasco et al. (74) found that SRBI levels in granulosa cells from 50 volunteers were correlated
with plasma levels of estradiol and the number of retrieved and
fertilized oocytes, although SR-BI levels were not correlated with
quality measurements of embryos, primary diagnosis of infertility,
or pregnancy outcomes. A study of 274 women by Yates et al. (75)
found no association of the SR-BI alleles tested with qualitative
measures of embryo viability (blastocyst number and grade).
However, 0 of 10 heterozygous carriers of one minor allele
(rs4238001) had viable fetuses after embryo transfer, raising the
possibility that this minor allele (and another allele in an African-
Materials and Methods
Pure C57BL/6 (Charles River Laboratories, Jackson Laboratory, and Taconic),
SR-BI−/−, SR-BI+/−, and SR-BI+/+ (5) mice were maintained on an RMH3000
laboratory diet ad libitum, and experiments were performed in concordance
with the guidelines from the committees for animal care at the Massachusetts Institute of Technology and Pontificia Universidad Católica de Chile (SI
Materials and Methods). Oocytes were collected from unprimed or primed
females, oocyte and follicle diameters were measured, in vitro maturation of
oocytes was performed, eggs were activated with SrCl2/EGTA, cholesterol
was depleted by MβCD or delivered by cholesterol/ MβCD complexes, relative
cholesterol content was assessed using filipin staining, eggs were imaged by
whole-mount staining and deconvolution microscopy, and statistical analyses were performed using standard procedures (SI Materials and Methods).
ACKNOWLEDGMENTS. We thank Helen Skaletsky for helpful suggestions,
Junmei Yao and Alan Discua for technical assistance, and Cindy Woolley
for help in preparing the manuscript. This work was supported by National
Institutes of Health (NIH) grants (to D.F.A. and M.K.), NIH Pre-Doctoral Training Grant T32GM007287 (to L.W.), an MIT International Science and Technology Initiatives Chile Cooperative Grant (to A.Y., D.B., A.R., and M.K.),
Chilean National Council for Scientific and Technological Research Program
Inserción en la Academia Grant 79090028 (to D.B.), Chilean National Council
for Scientific and Technological Research Program Fondo Nacional de Desarrollo Científico y Tecnológico Grants 11090064 and 1141236 (to D.B.) and
1110712 (to A.R.), and the Howard Hughes Medical Institute (D.C.P.).
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